2008 Research results

1. Comparison of 'Murasaki' and 'Koshu' Grape Cultivars Using Simple Sequence Repeat and Anthocyanin Analysis

Author: Nami GOTO-YAMAMOTO, Mineyo NUMATA, Toshi SHIMAMOTO, Ryosuke MOCHIOKA, and Akihiro HOSOMI
Abstract: It has been said that 'Murasaki' is an old native Japanese grape cultivar that is almost extinct, but also, contradictory to this, it has been stated that 'Murasaki' is in fact the same cultivar as 'Koshu'. At present, only one vine of 'Murasaki' is preserved in the Research Institute of Environment, Agriculture and Fisheries, Osaka Prefecture. This 'Murasaki' vine showed the same simple sequence repeat (SSR) profile as 'Koshu' for 26 SSR loci. Berry skin of 'Murasaki' showed slightly higher malvidin 3-glucoside concentration than that of 'Koshu' of the National Research Institute of Brewing in 2006. However, the anthocyanin concentration of 'Murasaki' was within the range of that of 'Koshu' from different locations, of different years, and probably of different clones. Thus, it appears that the conserved 'Murasaki' is the same as, or at least very similar to, 'Koshu'.
Source: J. ASEV Jpn., 19, 114-118 (2008)

2. Relationship between the concentrations of nitrogen/sulfur compounds in sakes before storing and those of polysulfides in sakes after storing

Author: Masaki Okuda, Atsuko Isogai, Midori Joyo, Nami Goto-Yamamoto and Shigeaki Mikami
Abstract: Using the sake samples presented at a sake contest in 2006, we investigated the relationships between concentrations of nitrogen/sulfur compounds before the storage of sake and concentrations of polysulfides (DMTS, DMDS) which are the main aroma compounds responsible for "hineka" in sake after the storage of sake, for a month at 50℃. The concentrations of total nitrogen in the sakes exhibited significantly positive correlations with the concentrations of sulfur compounds in the sakes. This result appeared to show that most of the sulfur compounds are derived from rice proteins. The ratio of sulfur of the amino acid formed against total sulfur averaged. 27.6% (min.17～max.45%) in the sake samples. After storing the sake samples, the concentrations of DMDS were the below detection threshold in all samples, and those of DMTS were above the detection threshold in half of the samples. The concentrations of polysulfides in the sakes after storing showed significant positive correlations with concentrations of sulfur compounds in the sakes before storing. Moreover, a partial correlation analysis showed that the concentration of amino acid containing sulfur had a significant positive correlation with that of polysulfide. These results suggest that sake containing high concentrations of sulfur compounds can produce a high concentration of polysulfide after storage for long periods.
Source: J. Brew. Soc. Japan, 104, 131-141 (2009)

3. The content of cadmium in rice, sake, and sake cake

Author: Kuniyasu GOTO
Abstract: The cadmium (Cd) content in 47 strains of rice for brewing sake, 25 commercial sakes, and 25 commercial sake cakes was measured. The samples were prepared by microwave digestion using HNO3 and H2O2, and then analyzed by an inductively coupled sequential plasma spectrometer (ICP-AES). The Cd content in brown rice, 70% polished rice, sake cake, and sake was 29.9, 25.3, 154.7 μg/kg and 1.29 μg/l, respectively. When the Cd content of brown rice was represented as 100, the relative content of the polished rice was 78 (average of 7 samples). These results showed that the Cd content was reduced slightly by the polishing process, and that the Cd in rice had hardly shifted to the sake, but shifted easily to the sake cake.
Source: J. Brew. Soc. Japan, 104, 209-214 (2009)

4. Ethanol stress stimulates the Ca2+-mediated calcineurin/Crz1 pathway in Saccharomyces cerevisiae

Author: Yoshio Araki, Hong Wu, Hiroshi Kitagaki, Takeshi Akao, Hiroshi Takagi and Hitoshi Shimoi
Abstract: Environmental stimuli elicit a stress response, which helps to maintain cell survival. In budding yeast Saccharomyces cerevisiae, environmental cues can activate calcineurin, a highly conserved Ca2⁺- and calmodulin-dependent protein phosphatase. Calcineurin dephosphorylates the transcription factor Crz1, leading to accumulation of Crz1 in the nuclei and expression of stress responsive genes under the control of a calcineurin-dependent response element (CDRE). Ethanol is the final product of sugar fermentation by yeast, and thus a frequently encountered yeast stressor. However, adaptation of yeast to ethanol stress is poorly understood. In this study, we show that ethanol stimulates calcineurin-dependent nuclear localization of Crz1 and CDRE-dependent gene expression. Moreover, cells in which CRZ1 is deleted exhibit defective adaptation to ethanol stress, while a multicopy plasmid of CRZ1 confers an increased level of adaptive stress tolerance to ethanol. Taken together, the results indicate that ethanol activates the calcineurin/Crz1 pathway and that CRZ1 is crucial for cell survival under ethanol-induced stress.
Source: J. Biosci. Bioeng., 107, 1-6 (2009)

5. Development of VNTR markers for three Aspergillus species used in brewing

Author: Kenta TOMIMURA, Kazuhiro IWASHITA, Osamu YAMADA, Ken KOBAYASHI
Abstract: Using a bioinformatics approach, we developed 18 variable number of tandem repeat markers for Aspergillus ozyzae for use in population genetic studies. Repeat sequences in the genome sequences of A. ozyzae were identified by a tandem repeat finding program. Length polymorphisms at 18 loci were examined in 41 strains of A. ozyzae. The total number of alleles per locus ranged from two to 20. Investigation of cross-species amplifications with A. sojae and A. tamarii showed success. The variable number of tandem repeat markers will be used to determine the population structure of these three Aspergillus species used in brewing.
Source: Molecular Ecology Resources, 9, 613-615 (2009)

6. Treatment and phosphorus removal from high-concentration organic wastewater by the yeast Hansenula anomala J224 PAWA.

Author: Watanabe T, Masaki K, Iwashita K, Fujii T, Iefuji H.
Abstract: A flocculent yeast, Hansenula anomala J224 PAWA, bred in this study, accumulated twice as much phosphorus as the wild type. Over a 30-d period, PAWA removed 70-80% of dissolved total phosphorus from sweet-potato and barley shochu wastewaters (alcoholic distillery wastewaters) while the wild type removed only 30%. Waste sludge was easily separated from effluent wastewater because PAWA cells made large flocks that rapidly settled. Component analysis suggested that PAWA sludge could be used as a protein source for feedstuff and as a phosphorus source for fertilizer. Under anaerobic conditions, denitrification was rapid, resulting in the removal of large amounts of nitrogen from barley shochu wastewater. These results suggest that small shochu manufacturers could benefit from using PAWA to remove phosphorus and organic compounds and then by using a combination of the upflow anaerobic sludge blanket and the downflow hanging sponge method (UASB-DHS method) for nitrification/denitrification.
Source: Bioresour Technol. 100, 1781-1785, (2009)

7. ONIOM Study of the Mechanism of the Enzymatic Hydrolysis of Biodegradable Plastics

Author: Yoshitake Sakae, Toshiaki Matsubara, Misako Aida, Hidemasa Kondo, Kazuo Masaki and Haruyuki Iefuji
Abstract: A cutinase-like enzyme (CLE), which is purified experimentally from the yeast Cryptococcus sp. strain S-2, has been recently found to degrade biodegradable plastics very efficiently. In this study, we theoretically examine the mechanism of hydrolysis of biodegradable plastics by the CLE by means of the ONIOM method. We optimize all the intermediates and the transition states involved in the considered enzymatic reaction and determine the energy surface of the entire catalytic cycle. The calculations show that the amino acid residues inside the pocket of the active site, Thr17 and Gln86, which stabilize the tetrahedral intermediates, and Gly115 in addition to Ser85, His180, and Asp165, which compose the catalytic triad, significantly contribute to the catalytic reaction. This is similar to the case of serine protease reported to date. Moreover, we have newly found that the energy barrier of the catalytic cycle is significantly lowered by the electronic effect of the OH group in the side-chain of Thr17 and bound water. The calculated potential energy surface of the reaction shows that the cleavage of the ester bond is a rate-determining step.
Source: Bulletin of the Chemical Society of Japan, 82, 338-346, (2009)

8. Characterization of an -L-rhamnosidase from Aspergillus kawachii and its gene

Author: Koseki T, Mese Y, Nishibori N, Masaki K, Fujii T, Handa T, Yamane Y, Shiono Y, Murayama T, Iefuji H.
Abstract: An -L-rhamnosidase was purified by fractionating a culture filtrate of Aspergillus kawachii grown on L-rhamnose as the sole carbon source. The α-l-rhamnosidase had a molecular mass of 90 kDa and a high degree of N-glycosylation of approximately 22%. The enzyme exhibited optimal activity at pH 4.0 and temperature of 50 ℃. Further, it was observed to be thermostable, and it retained more than 80% of its original activity following incubation at 60°C for 1 h. Its T₅₀ value was determined to be 72 ℃. The enzyme was able to hydrolyze -1,2- and -1,6-glycosidic bonds. The specific activity of the enzyme was higher toward naringin than toward hesperidin. The A. kawachii -L-rhamnosidase-encoding gene (Ak-rhaA) codes for a 655-amino-acid protein. Based on the amino acid sequence deduced from the cDNA, the protein possessed 13 potential N-glycosylation recognition sites and exhibited a high degree of sequence identity (up to 75%) with the -L-rhamnosidases belonging to the glycoside hydrolase family 78 from Aspergillus aculeatus and with hypothetical Aspergillus oryzae and Aspergillus fumigatus proteins.
Source: Appl Microbiol Biotechnol. 80, 1007-1013, (2008)

9. Screening and Identification of Precursor Compounds of Dimethyltrisulfide (DMTS) in Japanese Sake

Author: Atsuko Isogai, Ryoko Kanda, Yoshikazu Hiraga, Toshihide Nishimura, Hiroshi Iwata, and Nami Goto-Yamamoto
Abstract: Dimethyltrisulfide (DMTS) is involved in the unpalatable aroma of stale sake, called "hineka"; however, the mechanism underlying the formation of DMTS during the storage of sake has not been elucidated. Here we investigated the precursors of DMTS in sake. An experiment using [methyl-d3]-methionine showed that Strecker degradation of methionine plays a minor role in the formation of DMTS. Separation of components in sake by cation exchange resin revealed that DMTS precursors are present in the acidic/neutral fraction rather than in the basic one. Purification of the DMTS precursor compounds was carried out through several chromatographic steps, measuring DMTS producing potential as an index. High-resolution ESI-MS and 1-D/2-D NMR experiments enabled to identify one of the precursor compounds as 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one.
Source: Journal of Agricultural and Food Chemistry, 57, 189-195 (2009)

10. Brewing characteristics of haploid strains isolated from the sake yeast Kyokai no. 7

Author: Taku Katou, Hiroshi Kitagaki, Takeshi Akao and Hitoshi Shimoi
Abstract: Sake yeast exhibit various characteristics that make them more suitable for sake brewing compared to other yeast strains. Since sake yeast strains are Saccharomyces cerevisiae heterothallic diploid strains, it is likely that they have heterozygous alleles on homologous chromosomes (heterozygosity) due to spontaneous mutations. If this is the case, segregation of phenotypic traits in haploid strains after sporulation and concomitant meiosis of sake yeast strains would be expected to occur. To examine this hypothesis, we isolated 100 haploid strains from Kyokai no. 7 (K7), a typical sake yeast strain in Japan, and compared their brewing characteristics in small scale sake brewing tests. Analyses of the resultant sake samples showed a smooth and continuous distribution of analytical values for brewing characteristics, suggesting that K7 has multiple heterozygosities that affect brewing characteristics and that these heterozygous alleles do segregate after sporulation. Correlation and principal component analyses suggested that the analytical parameters could be classified into two groups indicating fermentation ability and the taste of sake.
Source: Yeast, 25, 799-807 (2008)

11. Bunch Shading During Different Developmental Stages Affects the Phenolic Biosynthesis in Berry Skins of 'Cabernet Sauvignon' Grapes

Author: Kazuya KOYAMA, Nami GOTO
Abstract: The effect of bunch shading during early development (before the onset of ripening) and/or during ripening on the phenolic composition of grape skins (Vitis vinifera L. cv. Cabernet Sauvignon) as well as on the mRNA levels of the biosynthetic genes on the flavonoid pathway was examined. Shading during early development resulted in decreased proanthocyanidin (PA) concentrations. The PA concentrations decreased during ripening, and the decrease of the concentrations was lower in berries shaded during early development than that in the exposed berries. Thus, no significant effect of shading during early development was observed at harvest. Shading during ripening did not influence this decline in the PAs. On the other hand, shading during early development induced changes in the composition such as a decrease of the trihydroxylated subunits within PAs, which agreed with the relative decrease of VvF3'5'H expression. The anthocyanin concentrations were remarkably reduced when the bunches were shaded during ripening, which was in accordance with the decreased transcription of several anthocyanin biosynthetic genes and transcriptional factors. Shading during early development did not influence the anthocyanin concentrations at harvest; however, it decreased the proportion of trihydroxylated anthocyanins. Thus, shading during early development also had an influence on the compounds biosynthesized during ripening.
Source: Journal of the American Society for Horticultural Science、133(6)、743-753、(2008)

12. Expression analysis of UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene in an interspecific hybrid grape between Vitis ficifolia var. ganebu and Vitis vinifera cv. Muscat of Alexandria

Author: Puspa Raj POUDEL, Nami GOTO-YAMAMOTO, Ryosuke MOCHIOKA, Ikuo KATAOKA, Kenji BEPPU
Abstract: Kadainou R-1, an interspecific hybrid grape derived from red (Vitis ficifolia var. ganebu) and white (V. vinifera cv. Muscat of Alexandria) grapes, accumulates high concentrations of anthocyanin in the berry skin. Hence, the expression of uridine 5'-diphosphate (UDP)-glucose:flavonoid 3-O-glucosyltransferase (UFGT), the key enzyme of the anthocyanin pathway, was examined in the berry skin of Kadainou R-1. As information on gene sequences of V. ficifolia var. ganebu and other wild grape species was unavailable, we performed GeneChip hybridization using biotin-labeled genomic deoxyribonucleic acid (DNA) to investigate how the genomic sequences of V. vinifera varieties and that of V. ficifolia var. ganebu differ. The study showed a lower correlation coefficient between V. vinifera cultivars and V. ficifolia var. ganebu than that among V. vinifera cultivars. The sequences of the UFGT gene derived from both parents of the red and white cultivars were sequenced in Kadainou R1 and revealed that both were expressed irrespective of the fact that it was not expressed in the white grape (male parent).
Source: Plant Biotechnol. Rep., 2, 233-238 (2008)

13. Investigation of Public Needs on Alcoholic Beverages

Author: Junichi KURAMITSU, Hitoshi UTSUNOMIYA, Katsumi HASHIZUME
Source: Report of the Research Institute of Brewing, 180, 57-72 (2008)

14. Investigation of consumers motives for their choice of sake, beer, and whisky

Author: Hitoshi UTSUNOMIYA, Katsumi HASHIZUME
Source: Report of the Research Institute of Brewing, 180, 73-92 (2008)

15. Relationship between sake cake ratios and atmospheric temperature conditions

Author: Katsumi HASHIZUME, Masaki OKUDA
Abstract: Correlation coefficients between the national mean data of sake cake ratios and mean data of monthly atmospheric temperatures of 21 meteorological observation points in Japan showed positive peaks in September, but did not show clear tendencies in winter. The results obtained suggest that atmospheric temperature conditions during the grain-filling period of material rice strongly affect the digestibility of steamed rice grains in sake mash. On the other hand, the effect of atmospheric temperature conditions during winter was not clearly observed.
Source: J. Brew. Soc. Japan, 103, 945-948 (2008)

16. Results of Sensory Evaluation and Analysis of the Western Type Alcoholic beverages Presented to the 45th contest

Author: S. Mikami, N. Goto, K. Koyama and J. Hiramatsu
Source: Report of the Research Institute of Brewing, 180, 17-31 (2008)

17. Analysis of Sake Component Presented to the Sake Contest in 2006

Author: H. Iwata, A. Isogai, S. Nakano, J. Hiramatsu
Source: Report of the Research Institute of Brewing, 180, 1-16 (2008)

18. Analysis of Traditional Shochu Presented to the 30th Contest in 2007

Author: S. Mikami, H. Iefuji, N. Mukai, J. Hiramatsu
Source: Report of the Research Institute of Brewing, 180, 32-42 (2008)

19. Analysis of Endocrine Disrupting Chemicals and Microbes in Alcoholic Beverages and Sake Cake

Author: K. Goto
Source: Report of the Research Institute of Brewing, 180, 43-56 (2008)

20. Effect of a Concentrate of Sake and Ethyl α-D-glucoside on Chronic Alcohol-induced Liver Injury in Mice

Author: Hanae IZU, Kazuhisa HIZUME, Kuniyasu GOTO, Masato HIROTSUNE
Abstract: We investigated the hepatoprotective effects of a concentrate of sake (CS) and a sake-specific sugar, ethyl α-D-glucoside (α-EG), against chronic alcohol-induced liver injury by measuring the plasma pyruvate transaminase (GPT) activity and the plasma and liver tryacylglycerol (TG) levels in mice. Male C57/BL6 mice were orally administered with CS (3 ml/kg) or α-EG (200 mg/kg) 5 times per week while on a Lieber-DeCarli diet with or without ethanol for 4 weeks. In the mice ingesting CS orα-EG, the alcohol-induced increase in plasma GPT activity and increases in plasma and liver TG levels were suppressed, suggesting that CS and α-EG suppress chronic alcohol-induced liver injury in mice. We showed that sake has components attenuating alcoholic liver injury.
Source: J. Brew. Soc. Japan, 103, 646-652 (2008)

21. Expression of multi-copy flavonoid pathway genes coincides with anthocyanin, flavonol and flavan-3-ol accumulation of grapevine

Author: S.T. Jeong, N. Goto-Yamamoto, K. Hashizume, M. Esaka
Abstract: The biosynthetic pathways of main grape flavonoids: anthocyanins, flavonols, and flavan-3-ols, hold in common the early step enzymes of biosynthetic pathway: chalcone synthase (CHS), chalcone isomerase (CHI), and flavanone 3-hydroxylase (F3H), the genes of which are multi-copied in the grape genome. The ratios of mRNA levels of the three Chss (Chs1, Chs2, and Chs3), as well as those of the two Chis (Chi1 and Chi2) and those of the two F3hs (F3h1 and F3h2), were different among organs, even though no organ-specificity was observed in the strictest sense. Multiple regression analysis demonstrated that the transcription of particular genes significantly coincided with the biosynthesis of a particular flavonoid: the transcription of Chi2 coincided with flavan-3-ol; Chs1, Chs2, F3h1 and F3h2 with flavonol; and Chs2, Chs3, and F3h2 with anthocyanin biosynthesis. Thus, the transcription of these multi-copy genes is likely induced differently for the biosyntheses of anthocyanins, flavonols, and flavan-3-ols.
Source: Vitis, 47, 135-140 (2008)

22. Breeding of wastewater treatment yeasts that accumulate high concentrations of phosphorus

Author: Watanabe T, Ozaki N, Iwashita K, Fujii T, Iefuji H.
Abstract: Inorganic phosphate is an essential nutrient. In general, microorganisms take up phosphorus when the extracellular phosphorus concentration is low, but not when it is high. In Saccharomyces cerevisiae, the major phosphate transporters, such as Pho84p, and acid phosphatases (APases), such as Pho5p, are regulated in parallel by the phosphate signal transduction pathway (PHO pathway). We found that PHO mutants expressing PHO84 and PHO5, even under high-P conditions, could take up phosphorus at twice the rate of the wild-type strain. The regulatory pathway for phosphorus accumulation in two wastewater treatment yeasts, Hansenula fabianii J640 and Hansenula anomala J224-1, was found to be similar to that in S. cerevisiae. We screened for mutants of these yeasts that constitutively expressed APase. Such mutants formed blue colonies on high phosphorus concentration agar plates containing 5-bromo-4-chloro-3-indolylphosphate (X-phosphate). We found four mutants of H. fabianii J640 and one mutant of H. anomala J224-1 that accumulated from 2.2 to 3.5 times more phosphorus than the parent strains. The growth rates and abilities to remove dissolved total nitrogen and dissolved organic carbon of the mutants were similar to those of the parent strains. In addition, the mutants removed 95% of dissolved total phosphorus from shochu wastewater, while the parent strain removed only 50%.
Source: Appl Microbiol Biotechnol. 80, 331-8 (2008)

23. Removal of triglyceride soil from fabrics by a novel lipase from Cryptococcus sp. S-2

Author: K. Thirunavukarasu, N.G. Edwinoliver, S. Durai Anbarasan, M.K. Gowthaman, H. Iefuji、 N.R. Kamini
Abstract: Removal of triglyceride molecules from fabrics could be accomplished in laundries using detergents containing lipases. The yeast, Cryptococcus sp. S-2 (CS2) produces a novel lipase, a cutinase-like enzyme that is compatible with various ionic, cationic and non-ionic surfactants as well as commercial detergents and oxidizing agents. In this study, the efficacy of CS2 lipase was assessed for the removal of triglyceride soil, like olive oil from fabrics in a single wash cycle using a tool of response surface methodology (RSM), so as to use the enzyme as an additive in laundry detergent formulations. A five-level four-factorial central composite rotatable design (CCRD) was used to evaluate the interactive effects of detergent concentration, lipase concentration, buffer pH and washing temperature on the percentage removal of triglyceride soil from cotton fabric. The model suggested that all the factors chosen had a significant impact on oil removal and the optimum conditions derived via RSM were 0.5% detergent, 1000 U of lipase, buffer pH of 8.0 and washing temperature of 37.8℃. Under optimal conditions, the removal of olive oil from cotton fabric was 19.6 and 43.1% higher at 20 and 37.8℃, respectively, in the presence of lipase,when compared to treatment with detergent wash alone. Moreover, the present work will also serve as a baseline of initial studies on cutinases and other related enzymes for its exploitation in laundry detergents.
Source: Process Biochemistry, 43, 701-706 (2008)

24. Two kinds of behavioral evaluation of intoxication by oral administration of ethanol in mice

Author: Hanae IZU, Kuniyasu GOTO, Shogo SAKATA
Abstract: Evaluation of ethanol intoxication in humans has difficulties in individual differences and ethical considerations. Intraperitoneal injection of ethanol has been used in evaluation of ethanol intoxication in nonhuman animals. We have developed two kinds of behavioral evaluations of intoxication by oral administration of ethanol in mice. Oral administration of ethanol (1.6～4.0 g/kg as a single dose, 0.8～1.6 g/kg dosed twice) increased both the number of open-arm entries and the time spent in the open arm of the elevated plus-maze test, demonstrating an anxiolytic-like effect of ethanol. Oral administration of ethanol (1.6 or 4.0 g/kg single dose, 1.2 or 2.4 g/kg dosed twice) significantly decreased locomotor activity under fasting conditions. Under semi-fasting conditions, oral administration of ethanol (1.6 g/kg single dose) transiently enhanced locomotor activity 1 h after administration but decreased subsequently for 48 h. Elevated plus maze and locomotor activity are useful for analyzing mild and mild-to-severe intoxicaton, respectively, caused by oral administration of ethanol on the condition used in this study.
Source: The Japanese Journal of Animal Psychology, 58, 1-13 (2008)

25. Aspergillus oryzae atfB encodes a transcription factor required for stress tolerance in conidia

Author: Kazutoshi Sakamoto, Toshi-hide Arima, Kazuhiro Iwashita, Osamu Yamada, Katsuya Gomi, Osamu Akita
Abstract: Using an Aspergillus oryzae EST database, we identified a gene encoding a transcription factor (atfB), which is a member of the ATF/CREB family. Expression of atfB was barely detectable during vegetative growth, but was readily detected during conidiation in solid-state culture. Microarray analyses showed that expression of many other genes, including catalase (catA), were downregulated in an atfB-disruptant. The expression of most of these genes was upregulated in the wild-type strain during the conidiation phase in solid-state culture, and the expression pattern was similar to that of atfB itself. In the absence of stress, e.g. heat shock or hydrogen peroxide, the conidial germination ratios for the ΔatfB strain and the wild-type strain were similar, but the stress tolerance of conidia carrying the ΔatfB deletion was less than that of the wild-type conidia. CRE-like DNA motifs, which are bound by ATF/CREB proteins, were found in the promoters of most of the downregulated genes in the ΔatfB strain. Thus, atfB appears to encode a transcription factor required for stress tolerance in conidia.
Source: Fungal Genetics and Biology 45(6), 922-932 (2008)

26. Common industrial sake yeast strains have three copies of the AQY1-ARR3 region of chromosome XVI in their genomes

Author: Ogihara, Hiroshi Kitagaki, Qian Wang, Hitoshi Shimoi
Abstract: Genomic analysis of industrial yeast strains is important for exploitation of their potential. We analysed the genomic structure of the most widely used sake yeast strain, Kyokai no. 7 (K7), by DNA microarray. Since the analysis suggested that the copy number of the AQY1-ARR3 region in the right arm of chromosome XVI was amplified, we performed Southern blot analysis using the AQY1 gene as a probe. The probe hybridized to three bands in the widely used sake strains derived from K7, but only to one band of 1.4 kb in the laboratory strains. Since the extra two bands were not observed in old sake strains, or in other industrial strains, the amplification of this region appeared to be specific for the widely used sake strains. The copy number of the AQY1-ARR3 region appeared to have increased by chromosomal translocation, since chromosomal Southern blot analysis revealed that the AQY1 probe hybridized to chromosomes IV and XIII, in addition to chromosome XVI, in which AQY1 of the laboratory strain is encoded. The chromosomal translocation was also confirmed by PCR analysis using primers that amplify the region containing the breakpoint. Cloning and sequencing of cosmids that encode the AQY1-ARR3 region revealed that this region is flanked by TG1-3 repeats on the centromere-proximal side in chromosomes IV and XIII, suggesting that amplification of this region occurred by homologous recombination through TG1-3 repeats. These results demonstrate the genomic characteristics of the modern widely used sake strains that discriminate them from other strains.
Source: Yeast, 25, 419-432 (2008)

27. Preliminary Study for the Development of a Long-Life, Continuous, Primary Fermentation System for Beer Brewing

Author: Takashi Inoue and Akihiro Mizuno
Abstract: A continuous primary bottom fermentation system for beer brewing with the ability to maintain a stable and high level of yeast activity during its long operation was designed and its performance was evaluated. The basic strategy was to realize the same physiologically or morphologically synchronized changes in yeast and wort constituents during the main fermentation as those realized in traditional bottom beer fermentations. The liquid nonabsorbent carrier particles used to fill the fermenter successfully promoted the fermentation of wort sugars, which were supplied from the top of the fermenter, by preventing their rapid sinking. In addition, the carrier particles displayed other benefits that result in effective operation. The quality of the harvested young beer was satisfactory, but had a low ester content that may be improved by further studies.
Source: J. Am. Soc. Brew. Chem. 66(2), 80-87 (2008)

28. Effect of Soy Peptide on Brewing Beer

Author: Sayuri KITAGAWA, Nobuhiko MUKAI, Yuko FURUKAWA, Kanako ADACHI, Akihiro MIZUNO, and Haruyuki IEFUJI
Abstract: We examined the effect of soy peptides (SPs) on the fermentation and growth of Yeast Bank Weihenstephan 34/70 (W34/70), a bottom-fermenting yeast. We compared fermentation for SP with that for a free amino acid (FAA) mixture having the same amino acid composition as SP, as a nitrogen source. Maltose syrup was used as a carbon source, and the medium contained excess amounts of essential minerals and vitamins. We observed that SP was better than FAA mixture at promoting fermentation and growth and that much more β-phenylethyl alcohol was produced during fermentation with SP than with FAA mixture. Subsequently, we compared fermentations with the FAA mixture and selected mixtures containing various dipeptides of Phe as a nitrogen source. We found that the rates of Phe metabolism and β-phenylethyl alcohol generation were much higher when Phe was presented as a dipeptide (Phe-Asp, Phe-Leu, or Phe-Phe) than when presented as FAA. These results show that amino acids such as Phe are absorbed more rapidly when presented as a peptide than as FAA, resulting in a more rapid production of β-phenylethyl alcohol.
Source: JOURNAL OF BIOSCIENCE AND BIOENGINEERING 105 (4), 360-366 (2008)

29. An acidic and thermostable carboxymethyl cellulase from the yeast Cryptococcus sp. S-2: Purification, characterization and improvement of its recombinant enzyme production by high cell-density fermentation of Pichia pastoris

Author: Jantaporn Thongekkaew, Hiroko Ikeda, Kazuo Masaki, Haruyuki Iefuji
Abstract: The extracellular carboxymethyl cellulase (CSCMCase) from the yeast, Cryptococcus sp. S-2, was produced when grown on cellobiose. It was purified to homogeneity from the supernatant by ultrafiltration, DEAE- 5PW anion exchange column and TSK-Gel G3000SW gel filtration. The purified enzyme was monomeric protein with molecular mass of approximately 34 kDa. The optimum temperature and pH for the action of the enzyme were at 40-50℃ and 3.5, respectively. It was stable at pH range of 5.5-7.5 and retained approximately 50% of its maximum activity after incubating at 90℃ for 1 h. Moreover, it could able to hydrolyze carboxymethyl cellulose sodium salt higher than insoluble cellulose substrate such as Avicel, SIGMACELL and CM cellulose. Due to its action at acidic pH and moderately stable at high temperature, the gene encoding carboxymethyl cellulase (CSCMCase) was isolated and improved the enzyme yield by high cell-density fermentation of Pichia pastoris. The CSCMCase cDNA contains 1023 nucleotides and encodes a 341-amino acid. It was successfully expressed under the control of alcohol oxidase I promoter using methanol induction of P. pastoris fermentation in a 2L ABLE bioreactor. The production of the recombinant carboxymethyl cellulases was higher than that from Cryptococcus sp. S-2 of 657-fold (2.75 and 4.2 x10^-3 mg protein L^-1, respectively) indicating that the leader sequence of CSCMCase has been recognized and processed as efficiently by P. pastoris. Furthermore, the recombinant enzyme was purified in two-step of ultrafiltration and hydrophobic interaction chromatography which would be much more convenient for large-scale purification for successful industrial application.
Source: Protein Expression and Purification 60, 140-146 (2008)

30. Inhibition of Mitochondrial Fragmentation during Sake Brewing Causes High Malate Production in Sake Yeast

Author: Hiroshi Kitagaki, Taku Kato, Atsuko Isogai, Shigeaki Mikami, Hitoshi Shimoi,
Abstract: We previously demonstrated the presence and fragmentation of mitochondria during alcohol fermentation. Here, we show that Fis1p induces mitochondrial fragmentation, and inhibition of mitochondrial fragmentation causes higher malate production during sake brewing. These findings indicate that mitochondrial morphology affects the metabolism of constituents, providing a breeding strategy for high-malate-producing yeasts.
Source: J. Biosci. Bioeng. 105(6), 675-678 (2008)